Mechanisms of Defective Hydroxylation of 5-Methylcytosine in MDS Include Pathways Other Than TET2 and IDH1/2

Abstract Abstract 462 In myelodysplastic syndrome (MDS), mutations in genes affecting epigenetic regulation constitute a link between genomic and epigenetic instability. Previously, we and others described mutations in TET2, coding for a 2-oxyglutarate-dependent methylcytosine dioxygenase, which converts 5-methycytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). Subsequently, dysfunction of wild type TET2 was mechanistically linked to neomorphic IDH mutations which deplete 2-oxyglutarate and produce a competitive inhibitor, 2-hydroxyglutarate. Previously, we established analytic tools to indirectly quantify 5-hmC content in leukemic genomes: in patients with myeloid malignancies 5-hmC levels are decreased as compared to healthy controls (p=1.8e-09). A decrease in 5-hmC levels correlated with dysfunction of TET2 as a consequence of inactivating hypomorphic mutations. Nevertheless, while in a majority of patients with decreased 5hmC levels TET2 mutations can be found, in a substantial minority of cases no explanation for the 5hmC deficiency has been found; down-modulation of TET2 mRNA and protein expression was absent and mutations in TET1 and TET3 have not been identified. Thus, other currently unidentified proteins may be directly or indirectly (via regulation of TET activity) involved in the deregulation of 5hmC levels in TET2 and IDH1/2-mutation-negative cases with low 5-hmC. To further investigate this issue we first characterized on a molecular levels patients with low 5-hmC using various approaches. SNP-A karyotyping failed to identify recurrent chromosomal defects in these patients that could point towards defects in pathogenic genes involved in the regulation of 5-hmC levels. We also screened 107 MDS patients to correlate of genomic 5-hmC content and the presence of recurrent mutations including IDH1/2, DNMT3A, ASXL1 and RUNX1 genes (as well as TET2). Within these genes, except for an association with TET2 mutations, a positive correlation with low 5-hmC levels was found only for IDH1/2 mutant cases (p=.05, n=5), whereas no correlation has been established for DNMT3A (p=.119, n=12), ASXL1 (p=.434, n=21) and RUNX1 (p=.602, n=22) mutant cases. While TET2 and IDH mutations were rarely seen together (n=1), none of the other studied gene mutations were mutually exclusive with TET2, suggesting contributions of defects in novel yet not identified genes. Several other genes similar to TET or IDH proteins, or hypothetically linked to DNA demethylation pathways could, at least theoretically, affect 5-hmC content, including for instance D2HGDH and the ELP gene family. However, no mutations were identified in these patients, except for identification of yet unknown SNPs in D2HGDH and ELP4 in some patients with unexplained low 5-hmC levels. In addition to the targeted approach we have also applied next generation sequencing technologies and sequenced whole exomes of malignant and non-affected cells (paired-end (2×100) Illumina HiSeq 2000) to identify novel acquired determinants of 5-mC hydroxymethylation in two representative patients. By using a selective algorithm, 18 overlapping potential somatic alterations in these patients were found in genes which could functionally affect 5-hmC content. In addition, several other mutated genes have been identified in each patient; these are being further investigated in other patients with low 5-hmC levels. Sanger sequencing was applied to confirm the presence of previously detected mutations in NF1 and KRAS, as well as all novel mutations, for instance in BRCC3 and SF3B1, in these patients. In sum, our results provide novel insights into the molecular mechanisms underlying MDS pathophysiology and describe the possibility that the TET family enzymes can act together with other putative proteins linked to DNA demethylation pathways. The use of high throughput sequencing technologies increase the probability of identification of novel changes which can be linked to functional consequences in these patients, ultimately furthering the understanding its role in genomic stability in MDS. Disclosures: No relevant conflicts of interest to declare.

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Recurrent Mutations Found In Acute Monocytic Leukemia by Exomes Sequencing

Blood ◽

10.1182/blood.v116.21.2490.2490 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2490-2490

Author(s):

Xiao-Jing Yan ◽

Jie Xu ◽

Zhao-Hui Gu ◽

Chun-Ming Pan ◽

Gang Lv ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prognostic Significance ◽

Point Mutations ◽

Gene Mutations ◽

Distinct Group ◽

Acute Monocytic Leukemia ◽

Monocytic Leukemia ◽

Genetic Changes ◽

Recurrent Mutations ◽

Acute Myelomonocytic Leukemia

Abstract Abstract 2490 Acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5), is a distinct group with characteristic clinical features and has been reported to have a poor prognosis. A subset of AML-M5 is associated with various chromosomal translocations involving the MLL locus at 11q23, while mutations are also reported in genes such as NPM1, FLT3, NRAS at different frequencies in this disease. However, these genetic changes occur only in a part of AML-M5 patients and some of them lack specificity due to the presence in other AML subtypes. To address the important genetic and molecular factors for the pathogenesis of M5 in a comprehensive manner, we sequenced exomes from nine AML-M5 initial bone marrow (BM) samples and matched control samples. We identified 64 somatic mutations within the coding sequences of 61 genes including 57 point mutations and 7 insertions or deletions (indels), among which 17 genes had at least 2 cases of mutations in 100 validated AML-M5 BM samples. We tested a part of these gene mutations in different subtypes of AML and found that some mutations are restricted to AML-M5 and AML-M4 (acute myelomonocytic leukemia) with more than 15% and 10% frequency, respectively. Thus, by systemic sequencing of exomes from a group of AML-M5 cases, we discovered recurring mutations that may play an essential role in the pathogenesis of AML with monocytic features.The prognostic significance of the mutations will be present in details. Disclosures: No relevant conflicts of interest to declare.

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Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽

10.1182/blood.v120.21.124.124 ◽

2012 ◽

Vol 120 (21) ◽

pp. 124-124

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yusuke Okuno ◽

Yuichi Shiraishi ◽

Yasunobu Nagata ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Conflicts Of Interest ◽

Massively Parallel Sequencing ◽

Gene Mutations ◽

Pediatric Leukemia ◽

Protein Coding ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML

Blood ◽

10.1182/blood.v122.21.3995.3995 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3995-3995

Author(s):

Arati V. Rao ◽

Yuri D. Fedoriw ◽

Kristy L. Richards ◽

Zhen Sun ◽

Cassandra L Love ◽

...

Keyword(s):

Exome Sequencing ◽

High Throughput Sequencing ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Cancer Genes ◽

Sequencing Data ◽

Illumina Platform ◽

Depth Analysis

Abstract Background Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML. Methodology We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform. Results We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs. Conclusion This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival. Disclosures: No relevant conflicts of interest to declare.

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A Novel Path for ATRA Differentiation Therapy in Acute Myeloid Leukemia with Isocitrate Dehydrogenase Mutations

Blood ◽

10.1182/blood.v124.21.3727.3727 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3727-3727

Author(s):

Jean-Emmanuel Sarry ◽

Helena Boutzen ◽

Christian Récher

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Granulocytic Differentiation ◽

Induced Differentiation ◽

Idh1 R132h ◽

Recurrent Mutations ◽

Idh Mutations ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by accumulation of malignant blasts with impaired differentiation programs due to recurrent mutations, among which IDH mutations occur in 15% of AML patients. These mutations lead to a block in erythroid commitment while they may also bias hematopoietic differentiation to myeloid lineage. Interestingly, Lyn tyrosine kinase is required for erythroid differentiation and we have observed a reduction of Lyn expression in the presence of IDH1-R132H mutation. It is also a negative regulator of ATRA-induced granulocytic differentiation. Accordingly, we hypothesized that IDH mutations may sensitize AML cells to ATRA-induced differentiation. Here, we report that clinically achievable doses of ATRA are sufficient to trigger differentiation specifically on AML cell lines, primary patient samples and xenograft mice models carrying IDH1 mutation as observed by an increase in CD11b expression, granulocytic enzyme activity and morphologic changes in May-Grunwald-Giemsa staining. We also showed that ATRA-induced terminal granulocytic differentiation increases apoptosis while decreases proliferation and colony formation specifically in IDH1 mutant cells. Moreover, inhibition of IDH1-R132H activity reduced ATRA-sensitivity while increasing expression of IDH mutation correlated with highest ATRA sensitivity. Furthermore, treatment with a cell-permeable form of the oncometabolite specifically produced by the mutant (eg. 2-HydroxyGlutarate) sensitized AML cells to ATRA-induced differentiation. Finally, because ATRA-induced differentiation triggers a transient increase of Lyn activation, its association with Lyn inhibitors synergistically increased ATRA-induced differentiation of IDH mutant blasts. In summary, our results showed that IDH mutations by producing 2-HG sensitized leukemic blasts to ATRA and that this synergizes with Lyn inhibition. Since 2HG concentration reaches millimolar in AML patient serum and is 100-fold higher in IDH mutated patients than in non-mutated ones, we would predict a strong efficacy and specificity of ATRA. Furthermore, as IDH mutations are systematically conserved at relapse, this therapeutic strategy might be promising to achieve a long-term remission specifically for this AML patient subgroup. Disclosures No relevant conflicts of interest to declare.

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Whole Exome Sequencing of Acute Myeloid Leukaemia Patients Identifies Somatic and Germline Mutations in Fanconi Anaemia Genes

Blood ◽

10.1182/blood.v124.21.698.698 ◽

2014 ◽

Vol 124 (21) ◽

pp. 698-698

Author(s):

Kyaw Zeya Maung ◽

James X Gray ◽

Paul J Leo ◽

Mahmoud Bassal ◽

Anna L Brown ◽

...

Keyword(s):

Dna Repair ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Gene Mutations ◽

Research Network ◽

Fanconi Anaemia ◽

Molecular Characteristics ◽

Whole Exome ◽

Recurrent Mutations

Abstract Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Uncover TET1 Targets in MLL -Rearranged Leukemia

Blood ◽

10.1182/blood.v126.23.3632.3632 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3632-3632

Author(s):

Hengyou Weng ◽

Huilin Huang ◽

He Huang ◽

Okwang Kwon ◽

Ping Chen ◽

...

Keyword(s):

Myeloid Leukemia ◽

Molecular Mechanisms ◽

Isotope Labeling ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Dna Demethylation ◽

In Vivo Models ◽

A Genome ◽

Wide Range

Abstract 5-hydroxymethylcytosine (5hmC), also called the "sixth DNA base", is involved in the DNA demethylation process which generally leads to gene activation. Formation of 5hmC is catalyzed by the Ten-Eleven-Translocation (TET) family proteins, with TET1 being the founding member. The expression of TET1 protein and the global level of its enzymatic product, 5hmC, is markedly reduced in a wide range of solid tumors, including melanoma, prostate, breast, lung, and liver cancer, suggesting that TET1 functions as a tumor suppressor in these types of cancers. However, a recent study from our group demonstrated that TET1 expression and the associated 5hmC levels are significantly up-regulated in MLL -rearranged leukemia, revealing the oncogenic role of TET1 in this type of acute myeloid leukemia (AML) (Huang H, et al. PNAS 2013; 110(29):11994-9). In support of this, another study from a different group showed that high 5hmC level is an independent predictor of poor overall survival in patients with AML (Kroeze LI, et al. Blood 2014; 124(7):1110-8). However, how TET1, as a critical methylcytosine dioxygenase, plays its oncogenic role in AML, especially in MLL -rearranged leukemia, is still unclear. To address this issue, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. When TET1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, 123 proteins were found downregulated whereas 191 were upregulated with a fold-change cutoff of 1.2. The expression changes of a set of these genes were confirmed by quantitative PCR in MLL-ENL-ERtm cells and mice samples with TET1 knock-down or depletion. After taking into account the correlation of TET1 and its candidate targets in several sets of AML patient samples, we focused on IDH1 and PSIP1, which represent the negatively- and positively-regulated targets by TET1, respectively. IDH1 encodes an isocitrate dehydrogenase whose mutations are frequently found in AML, whereas the PSIP1 protein is shown to be required for both MLL-dependent transcription and leukemic transformation. Chromatin immunoprecipitation (CHIP) assays suggest that TET1 directly binds to the CpG islands in the promoters of these two genes. Forced expression of Idh1 in leukemic bone marrow cells collected from mice developed MLL-AF9-driven AML significantly inhibited the colony-forming capacity of these cells, which mimics the effect of TET1 knock-out. We are now further investigating the functions and underlying molecular mechanisms of IDH1 and PSIP1 in AML using both in vitro and in vivo models. Considering the important roles of IDH1 and PSIP1 in AML, our findings will provide new insight into the mechanisms underlying the oncogenic role of TET1 in MLL -rearranged leukemia and may ultimately lead to the development of targeted therapy of AML. Disclosures No relevant conflicts of interest to declare.

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Genetic Basis of Myeloid Proliferation Related to Down Syndrome

Blood ◽

10.1182/blood.v120.21.535.535 ◽

2012 ◽

Vol 120 (21) ◽

pp. 535-535

Author(s):

Kenichi Yoshida ◽

Tsutomu Toki ◽

Myoung-ja Park ◽

Yusuke Okuno ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Down Syndrome ◽

Exome Sequencing ◽

High Throughput Sequencing ◽

Somatic Mutations ◽

Gene Mutations ◽

Whole Genome ◽

Discovery Cohort ◽

Whole Exome ◽

Recurrent Mutations ◽

Gata1 Mutation

Abstract Abstract 535 Background Transient abnormal myelopoiesis (TAM) represents a self-limited proliferation exclusively affecting perinatal infants with Down syndrome (DS), morphologically and immunologically characterized by immature blasts indistinguishable from acute megakaryoblastic leukemia (AMKL). Although spontaneous regression is as a rule in most cases, about 20–30% of the survived infants develop non-self-limited AMKL (DS-AMKL) 3 to 4 years after the remission. As for the molecular pathogenesis of these DS-related myeloid proliferations, it has been well established that GATA1 mutations are detected in virtually all TAM cases as well as DS-AMKL. However, it is still open to question whether a GATA1 mutation is sufficient for the development of TAM, what is the cellular origin of the subsequent AMKL, whether additional gene mutations are required for the progression to AMKL, and if so, what are their gene targets, although several genes have been reported to be mutated in occasional cases with AMKL, including JAK2/3, TP53 and FLT3. Methods To answer these questions, we identify a comprehensive spectrum of gene mutations in TAM/AMKL cases using whole genome sequencing of three trio samples sequentially obtained at initial presentation of TAM, during remission and at the subsequent relapse phase of AMKL. Whole exome sequencing was also performed for TAM (N=16) and AMKL (N=15) samples, using SureSelect (Agilent) enrichment of 50M exomes followed by high-throughput sequencing. The recurrent mutations in the discovery cohort were further screened in an extended cohort of DS-AMKL (N = 35) as well as TAM (N = 26) and other AMKL cases (N = 19) using target deep sequencing. Results TAM samples had significantly fewer numbers of somatic mutations compared to AMKL samples with the mean numbers of all mutations of 30 (1.0/Mb) and 180 (6.0/Mb) per samples in whole genome sequencing or non-silent somatic mutations of 1.73 and 5.71 per sample in whole exome sequencing in TAM and AMKL cases, respectively (p=0.001). Comprehensive detections of the full spectrum of mutations together with subsequent deep sequencing of the individual mutations allowed to reveal more complicated clonological pictures of clonal evolutions leading to AMKL. In every patient, the major AMKL clones did not represent the direct offspring from the dominant TAM clone. Instead, the direct ancestor of the AMKL clones could be back-traced to a more upstream branch-point of the evolution before the major TAM clone had appeared or, as previously reported, to an earlier founder having an independent GATA1 mutation. Intratumoral heterogeneity was evident at the time of diagnosis as the presence of major subpopulations in both TAM and AMKL populations, which were more often than not characterized by RAS pathway mutations. While GATA1 was the only recurrent mutational target in the TAM phase, 8 genes were recurrently mutated in AMKL samples in whole genome/exome sequencing, including NRAS, TP53 and other novel gene targets that had not been previously reported to be mutated in other neoplasms. The recurrent mutations found in the discovery cohort, in addition to known mutational targets in myeloid malignancies, were screened in an extended cohort of DS-associated myeloid disorders (N=61) as well as other AMKL cases, using high-throughput sequencing of SureSelect-captured and/or PCR amplified targets. Secondary mutations other than GATA1 mutations were found in 3 out of 26 TAM, 20 out of 35 DS-AMKL and 4 out of 19 other AMKL cases. Conclusion TAM is characterized by a paucity of somatic mutations and thought to be virtually caused by a GATA1 mutation in combination with constitutive trisomy 21. Subsequent AMKL evolved from a minor independent subclone acquiring additional mutations. Secondary genetic hits other than GATA1 mutations were common, where deregulated epigenetic controls as well as abnormal signaling pathway mutations play a major role. Disclosures: No relevant conflicts of interest to declare.

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Whole-Exome Resequencing Identifies Somatic Mutations Of BCOR and BCORL1 Transcriptional Corepressor Genes and Major Cohesin Complex Component Genes In Pediatric Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.834.834 ◽

2013 ◽

Vol 122 (21) ◽

pp. 834-834

Author(s):

Norio Shiba ◽

Kenichi Yoshida ◽

Yasunobu Nagata ◽

Ayana Kon ◽

Yusuke Okuno ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Somatic Mutations ◽

Conflicts Of Interest ◽

Gene Mutations ◽

Transcriptional Corepressor ◽

Whole Exome ◽

Recurrent Mutations ◽

Pediatric Aml ◽

Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Currently, targeted sequencing efforts have identified several mutations that carry diagnostic and prognostic information such as RAS, KIT, and FLT3 in both adult and pediatric AML, and NPM1 and TET2 in adult AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be enabled the discovery of recurrent mutations in IDH1/2, and DNMT3A in adult AML. However, these mutations are extremely rare in pediatric AML. Methods To reveal a complete registry of gene mutations and other genetic lesions, whole-exome resequencing of paired tumor-normal DNA from 19 cases were analyzed with a mean coverage of approximately x100, and 82 % of the target sequences were analyzed at more than x20 depth on average. We selected various cases in age, FAB classification and karyotypes, including 5 cases with core-binding-factor AML, 6 cases with MLL-rearrangement and 2 acute megakaryoblastic leukemia cases. Results and Discussion A total of 80 somatic mutations or 4.2 mutations per sample were identified. As the mean number of somatic mutations reported in adult AML was about ten, somatic mutations in pediatric AML might be fewer than in adult AML. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BRAF, BCORL1, DAZAP1, CUL2, ASXL2, MLL2, MLL3, SMC3 and RAD21. Among these, what immediately drew our attention were SMC3 and RAD21, because they belong to the major cohesin components. Cohesin is a multimeric protein complex conserved across species and composed of four core subunits, i.e., SMC1, SMC3, RAD21, and STAG proteins, forming a ring-like structure. Cohesin is engaged in cohesion of sister chromatids during cell division, post-replicative DNA repair, and regulation of global gene expression through long-range cis-interactions. Furthermore, we also drew our attention to BCORL1, because it is a transcriptional corepressor, and can bind to class II histone deacetyllases (HDAC4, HDAC5, HDAC7), to interact with the CTBP1 corepressor, and to affect the repression of E-cadherin. BCOR is also a transcriptional corepressor and play a key role in the regulation of early embryonic development, mesenchymal stem cell function and hematopoiesis. To confirm and extend the initial findings in the whole-exome sequencing, we studied mutations of the above 8 genes, in pediatric AML (N = 190) using a high-throughput mutation screen of pooled DNA followed by confirmation/ identification of candidate mutations. In total, 32 mutations were identified in 31 of the 190 specimens of pediatric AML [BCOR (N = 7), BCORL1 (N = 7), RAD21 (N = 7), SMC3 (N = 5), SMC1A (N = 1), and STAG2 (N = 3)]. The mutually exclusive pattern of the mutations in these BCOR, BCORL1 and cohesin components genes was confirmed in this large case series, suggesting a common impact of these mutations on the pathogenesis of pediatric AML. The 4-year overall survival of these cases with major cohesin components gene mutations was relatively favorable (12/16 or 75.0%), but the outcome of cases with BCOR or BCORL1 cases was unfavorable (8/14 or 57.1%). Conclusion Whole exome resequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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Landscape and dynamics of transcription initiation in the malaria parasitePlasmodium falciparum

10.1101/024356 ◽

2015 ◽

Author(s):

Sophie Adjalley ◽

Christophe Chabbert ◽

Bernd Klaus ◽

Vicent Pelechano ◽

Lars Steinmetz

Keyword(s):

Transcription Initiation ◽

Molecular Mechanisms ◽

High Throughput Sequencing ◽

Regulation Of Gene Expression ◽

Nucleotide Composition ◽

Transcriptional Unit ◽

Developmental Cycle ◽

Transcription Start Sites ◽

Sequencing Technologies ◽

Nucleotide Resolution

The lack of a comprehensive map of transcription start sites (TSS) across the highly AT-rich genome ofP. falciparumhas hindered progress towards deciphering the molecular mechanisms that underly the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single nucleotide-resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcriptional unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.

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Multi-omics annotation of human long non-coding RNAs

Biochemical Society Transactions ◽

10.1042/bst20191063 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1545-1556 ◽

Cited By ~ 2

Author(s):

Qianpeng Li ◽

Zhao Li ◽

Changrui Feng ◽

Shuai Jiang ◽

Zhang Zhang ◽

...

Keyword(s):

Human Genome ◽

Functional Annotation ◽

Molecular Mechanisms ◽

High Throughput Sequencing ◽

Rna World ◽

Omics Data ◽

Biological Processes ◽

Sequencing Technologies ◽

Non Coding Rnas ◽

Powerful Strategy

LncRNAs (long non-coding RNAs) are pervasively transcribed in the human genome and also extensively involved in a variety of essential biological processes and human diseases. The comprehensive annotation of human lncRNAs is of great significance in navigating the functional landscape of the human genome and deepening the understanding of the multi-featured RNA world. However, the unique characteristics of lncRNAs as well as their enormous quantity have complicated and challenged the annotation of lncRNAs. Advances in high-throughput sequencing technologies give rise to a large volume of omics data that are generated at an unprecedented rate and scale, providing possibilities in the identification, characterization and functional annotation of lncRNAs. Here, we review the recent important discoveries of human lncRNAs through analysis of various omics data and summarize specialized lncRNA database resources. Moreover, we highlight the multi-omics integrative analysis as a powerful strategy to efficiently discover and characterize the functional lncRNAs and elucidate their potential molecular mechanisms.

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